The present invention relates to improvements in the diagnosis of metastic disease. In particular, the invention relates to techniques for detecting the presence of proteolytic enzymes associated with metastatic disease.
Although a cure for most forms of cancer remains elusive, early detection and treatment have historically provided the best prognosis. Considerable effort over the years has been directed to developing diagnostic tests which give an indication of the presence of metastatic disease at an early stage.
Recently, a relationship between matrix metalloproteinases (MMP's) and metastasis has been suggested. MMP's are a family of closely related metal-dependent endopeptidases secreted by mesenchymal cells. For example, gelatinase A (MMP-2 or 72 kDa gelatinase/type IV collagenase) and gelatinase B (MMP-9 or 92 kDa gelatinase/type IV collagenase) have been identified as playing a major role in cancer invasion and metastasis. Stromelysin and PUMP (Putative Metalloproteinase), metalloproteinases with broader substrate specificity, are also thought to participate in the metastatic process. Three of these metalloproteinases have been identified in mammalian plasma.
Metastasis is a complex sequence of events in which malignant cells detach and disseminate from a primary tumor. The malignant cells invade adjacent tissue, penetrate into blood and lymphatic vessels, circulate to distant sites and eventually attach to and penetrate tissues in distant organs, thereby proliferating the malignancy. An important part of the metastatic process is the degradation of extracellular basement membranes by various proteolytic enzymes. Basement membranes are collagen-containing connective tissues which form a tough continuous sheet and separate the various cell layers such as the epithelial, endothelial and parenchymal cells from interstitial connective tissue.
Proteolytic enzymes associated with the metastatic process are found circulating in the form of activated enzyme (free enzyme), latent free enzyme and enzyme complexed with endogenous proteinase inhibitors. Only the active forms, however, digest the connective tissue substrates. The body's own natural defense mechanisms rapidly inactivate MMP's by compiexing the enzymes with specific tissue inhibitors of metalloproteinases (TIMP's). Such inhibitors found in the tissues and circulating in the plasma include TIMP-1, TIMP-2 and also alpha-2 macroglobulin which is primarily in blood.
In spite of the relationship between elevated levels of certain destructive proteases and the presence of metastatic disease, it has been difficult to use the relationship to provide an accurate indication of metastatic disease. In the past, it has only been possible to detect free or activated forms of the metalloproteinases, leaving a substantial portion of the inactivated enzymes complexed with inhibitors undetected. Recently it has been shown, however, that latent gelatinase A forms complexes with TIMP-2 and latent gelatinase B forms complexes with TIMP-1. Thus, these gelatinases can exist outside the cell in complexed forms with TIMPs.
U.S. Pat. No. 4,677,058 discloses purifying and detecting type IV collagenase antigens from malignant tumor cells. Similarly, U.S. Pat. No. 4,808,528 discloses antibodies specific to type IV collagenase enzyme antigens. U.S. Pat. No. 4,816,400, a division of the '058 U.S. Pat. No., supra, discloses immunological determination of type IV collagenase antigens using polyclonal and monoclonal antibodies. None of these references, however, disclose detecting collagenase enzyme-inhibitor complexes associated with metastatic disease.
In spite of the investigation of the role of metalloproteinases in metastatic disease, the total amount of MMP's being released and thus the actual metastatic activity has not been detectable. Indeed, due to the usually rapid inactivation of activated MMP's by TIMP's, measuring only free, circulating MMP's would fail to indicate the presence of many underlying diseases. Determining whether or not the complexes formed between MMP's and TIMP's form more reliable and sensitive diagnostic tests has yet to be investigated.
It is therefore an object of the present invention to provide highly specific and reliable diagnostic agents and methods to determine the presence of metastatic disease based on measuring matrix metalloproteinase inhibitor complex levels.
Other and further objects and advantages of the present invention will become apparent to those skilled in the art from a consideration of the following description taken together with the accompanying figure.